大黄鱼microRNA荧光定量PCR中内参基因的选择与评价

为筛选适用于大黄鱼miRNA研究的内参基因,选取饥饿试验组(EG)中饥饿21d及正常投喂组(CG,对照)的大黄鱼肌肉组织进行miRNA高通量测序,初步筛选得到稳定表达的6个miRNA:miR-23a-3p,miR-4508,miR-1961,miR-1297,miR-1956-3p和Let-7。采用实时荧光定量PCR法检测这6个miRNA和3个传统的内参基因(U6,5S和18S)在大黄鱼6种组织(肌肉、肝脏、肾、脾、脑和心脏)以及不同饥饿时间段的肌肉组织中的表达,并利用Ge Norm,Best Keeper以及Norm Finder对数据进行分析。结果表明,miRNA的表达稳定性优于传统内参基因。在CG组大黄鱼不同组织中,最稳定单内参基因是miR-23a-3p,最佳内参基因组合为miR-23a-3p和Let-7。在不同饥饿时间的EG组大黄鱼肌肉组织中,最稳定单内参基因是miR-23a-3p,最佳内参基因组合为Let-7和miR-1961。本试验结果为研究大黄鱼miRNA时内参基因的选择提供了参考。

Selection and Evaluation of Reference Genes in Quantitative PCR Analysis of microRNA in Larimichthys crocea

In order to screen the appropriate reference genes for the quantitative PCR(qPCR) used in the microRNA (miRNA) analysis of Larimichthys crocea,muscle tissues of Larimichthys croscea from the fasting 21-day individuals (fasting experimental group,EG) and normal feeding individuals (control group,CG) were collected respectively.Their microRNAs were sequenced with Hiseq technique,and six stable expressed microRNAs,miR-23a-3p,miR-4508,miR-1961,miR-1297,miR-1956-3p and Let-7,were obtained.Expression levels of the six microRNAs and three traditional reference genes (U6,5S and 18S) were examined by quantitative PCR method in six different tissues (muscle,liver,kidney,spleen,brain and heart) of normal feeding Larimichthys crocea and in the muscle tissues of Larimichthys crocea at different fasting periods.Experimental data were analyzed by GeNorm,BestKeeper and NormFinder.The results showed that expression stability of microRNAs was better than that of three traditional reference genes.In the different tissues of CG individuals,the most stable single reference gene was miR-23a-3p,and the best combination of reference genes was miR-23a-3p and Let-7.In the muscle tissues of EG individuals with different fasting periods,the most stable single reference gene was also miR-23a-3p while the best combination of reference genes was Let-7 and miR-1961.These results provide useful information on selecting suitable reference gene in microRNA research in Larimichthys crocea.